Functional genomics screens reveal a role for TBC1D24 and SV2B in antibody-dependent enhancement of dengue virus infection.

Dengue virus (DENV) can hijack non-neutralizing IgG antibodies to facilitate its uptake into target cells expressing Fc gamma receptors (FcgR) - a process known as antibody-dependent enhancement (ADE) of infection. Beyond a requirement for FcgR, host dependency factors for this non-canonical infection route remain unknown. To identify cellular factors exclusively required for ADE, here, we performed CRISPR knockout screens in an in vitro system permissive to infection only in the presence of IgG antibodies. Validating our approach, a top hit was FcgRIIa, which facilitates binding and internalization of IgG-bound DENV but is not required for canonical infection. Additionally, we identified host factors with no previously described role in DENV infection, including TBC1D24 and SV2B, both of which have known functions in regulated secretion. Using genetic knockout and trans-complemented cells, we validated a functional requirement for these host factors in ADE assays performed with monoclonal antibodies and polyclonal sera in multiple cell lines and using all four DENV serotypes. We show that knockout of TBC1D24 or SV2B impaired binding of IgG-DENV complexes to cells without affecting FcgRIIa expression levels. Thus, we identify cellular factors beyond FcgR that are required for ADE of DENV infection. Our findings represent a first step towards advancing fundamental knowledge behind the biology of ADE that can ultimately be exploited to inform vaccination and therapeutic approaches.

An RNA-centric dissection of host complexes controlling flavivirus infection.

Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.

Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus.

Encephalomyocarditis virus (EMCV) is an animal pathogen and an important model organism, whose receptor requirements are poorly understood. Here, we employed a genome-wide haploid genetic screen to identify novel EMCV host factors. In addition to the previously described picornavirus receptors sialic acid and glycosaminoglycans, this screen unveiled important new host factors for EMCV. These factors include components of the fibroblast growth factor (FGF) signaling pathway, such as the potential receptors FGFR1 and ADAM9, a cell-surface metalloproteinase. By employing various knockout cells, we confirmed the importance of the identified host factors for EMCV infection. The largest reduction in infection efficiency was observed in cells lacking ADAM9. Pharmacological inhibition of the metalloproteinase activity of ADAM9 did not affect virus infection. Moreover, reconstitution of inactive ADAM9 in knockout cells restored susceptibility to EMCV, pointing to a proteinase-independent role of ADAM9 in mediating EMCV infection. Using neutralization assays with ADAM9-specific antiserum and soluble receptor proteins, we provided evidence for a role of ADAM9 in EMCV entry. Finally, binding assays showed that ADAM9 facilitates attachment of EMCV to the cell surface. Together, our findings reveal a role for ADAM9 as a novel receptor or cofactor for EMCV.IMPORTANCE EMCV is an animal pathogen that causes acute viral infections, usually myocarditis or encephalitis. It is thought to circulate mainly among rodents, from which it is occasionally transmitted to other animal species, including humans. EMCV causes fatal outbreaks of myocarditis and encephalitis in pig farms and zoos, making it an important veterinary pathogen. Although EMCV has been widely used as a model to study mechanisms of viral disease in mice, little is known about its entry mechanism. Here, we employ a haploid genetic screen for EMCV host factors and identify an essential role for ADAM9 in EMCV entry.

Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors.

The oligosaccharyltransferase (OST) is a multisubunit enzyme complex that N-glycosylates proteins in the secretory pathway and is considered to be constitutive and unregulated. However, small-molecule OST inhibitors such as NGI-1 provide a pharmacological approach for regulating N-linked glycosylation. Herein we design cell models with knockout of each OST catalytic subunit (STT3A or STT3B) to screen the activity of NGI-1 and its analogs. We show that NGI-1 targets the function of both STT3A and STT3B and use structure-activity relationships to guide synthesis of catalytic subunit-specific inhibitors. Using this approach, pharmacophores that increase STT3B selectivity are characterized and an STT3B-specific inhibitor is identified. This inhibitor has discrete biological effects on endogenous STT3B target proteins such as COX2 but does not activate the cellular unfolded protein response. Together this work demonstrates that subsets of glycoproteins can be regulated through pharmacologic inhibition of N-linked glycosylation.

A Small-Molecule Oligosaccharyltransferase Inhibitor with Pan-flaviviral Activity.

The mosquito-borne flaviviruses include important human pathogens such as dengue, Zika, West Nile, and yellow fever viruses, which pose a serious threat for global health. Recent genetic screens identified endoplasmic reticulum (ER)-membrane multiprotein complexes, including the oligosaccharyltransferase (OST) complex, as critical flavivirus host factors. Here, we show that a chemical modulator of the OST complex termed NGI-1 has promising antiviral activity against flavivirus infections. We demonstrate that NGI-1 blocks viral RNA replication and that antiviral activity does not depend on inhibition of the N-glycosylation function of the OST. Viral mutants adapted to replicate in cells deficient of the OST complex showed resistance to NGI-1 treatment, reinforcing the on-target activity of NGI-1. Lastly, we show that NGI-1 also has strong antiviral activity in primary and disease-relevant cell types. This study provides an example for advancing from the identification of genetic determinants of infection to a host-directed antiviral compound with broad activity against flaviviruses.

Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling.

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling ß-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the ß-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems.

Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens.

The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells.

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.

The adherens junctions control susceptibility to Staphylococcus aureus α-toxin.

Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

Kinetic pathway of 40S ribosomal subunit recruitment to hepatitis C virus internal ribosome entry site.

Translation initiation can occur by multiple pathways. To delineate these pathways by single-molecule methods, fluorescently labeled ribosomal subunits are required. Here, we labeled human 40S ribosomal subunits with a fluorescent SNAP-tag at ribosomal protein eS25 (RPS25). The resulting ribosomal subunits could be specifically labeled in living cells and in vitro. Using single-molecule Förster resonance energy transfer (FRET) between RPS25 and domain II of the hepatitis C virus (HCV) internal ribosome entry site (IRES), we measured the rates of 40S subunit arrival to the HCV IRES. Our data support a single-step model of HCV IRES recruitment to 40S subunits, irreversible on the initiation time scale. We furthermore demonstrated that after binding, the 40S:HCV IRES complex is conformationally dynamic, undergoing slow large-scale rearrangements. Addition of translation extracts suppresses these fluctuations, funneling the complex into a single conformation on the 80S assembly pathway. These findings show that 40S:HCV IRES complex formation is accompanied by dynamic conformational rearrangements that may be modulated by initiation factors.

Differential virus host-ranges of the Fuselloviridae of hyperthermophilic Archaea: implications for evolution in extreme environments.

An emerging model for investigating virus-host interactions in hyperthermophilic Archaea is the Fusellovirus-Sulfolobus system. The host, Sulfolobus, is a hyperthermophilic acidophile endemic to sulfuric hot springs worldwide. The Fuselloviruses, also known as Sulfolobus Spindle-shaped Viruses (SSVs), are "lemon" or "spindle"-shaped double-stranded DNA viruses, which are also found worldwide. Although a few studies have addressed the host-range for the type virus, Sulfolobus Spindle-shaped Virus 1 (SSV1), using common Sulfolobus strains, a comprehensive host-range study for SSV-Sulfolobus systems has not been performed. Herein, we examine six bona fide SSV strains (SSV1, SSV2, SSV3, SSVL1, SSVK1, SSVRH) and their respective infection characteristics on multiple hosts from the family Sulfolobaceae. A spot-on-lawn or "halo" assay was employed to determine SSV infectivity (and host susceptibility) in parallel challenges of multiple SSVs on a lawn of a single Sulfolobus strain. Different SSVs have different host-ranges with SSV1 exhibiting the narrowest host-range and SSVRH exhibiting the broadest host range. In contrast to previous reports, SSVs can infect hosts beyond the genus Sulfolobus. Furthermore, geography does not appear to be a reliable predictor of Sulfolobus susceptibility to infection by any given SSV. The ability for SSVs to infect susceptible Sulfolobus host does not appear to change between 65°C and 88°C (physiological range); however, very low pH appears to influence infection. Lastly, for the virus-host pairs tested the Fusellovirus-Sulfolobus system appears to exhibit host-advantage. This work provides a foundation for understanding Fusellovirus biology and virus-host coevolution in extreme ecosystems.